Immunoconjugates are covalently bonded hybrid molecules composed of a recognition portion, such as an antibody molecule, an antibody fragment, or a functional equivalent thereof, and a biologically active portion, such as a toxin, a toxin fragment, a drug, a biologic response modifier, or a radioisotope. Immunoconjugates have enormous potential as potent anti-tumor agents, due to the selectivity imparted to the hybrid molecules by the antibody portion of the immunoconjugate. The exquisite selectivity of antibodies or antibody fragments permits delivery of increased doses of cytotoxic, inhibitory or radiolabeled moieties to a defined population of cells.
Originally, immunoconjugates were formed using polyclonal antibodies coupled to toxin molecules. Recently, the development of hybridoma technology has made available monoclonal antibodies that bind to a specific epitope of an antigen. This is in contrast to polyclonal antibodies that bind to multiple antigens or epitopes. Although monoclonal antibodies or fragments thereof offer improved specificity and reproducibility of a given hybrid molecule, certain technical problems in the preparation of immunoconjugates have been recognized.
For example, the linkage of antibody to toxin is one variable that has been examined by several investigators. In general, immunoconjugates may be formed by linking two molecules through disulfide bonds, which can be reductively cleaved, or by linking through bonds that are not affected by reducing agents, such as amide or thioether bonds.
Intact toxin molecules, such as diphtheria toxin, ricin, and abrin, are composed of an A chain and a B chain linked by a disulfide bond. The B chain of ricin binds to specific receptors on the surface of target cells, and is believed to participate in the internalization of the A chain. The A chain contains the biologically active portion of the toxin molecule. Upon reduction of the disulfide bond between the A and B chains, the A chain is released into the cytoplasm and participates in a biochemical reaction that results in inhibition of protein synthesis in target cells. Therefore, it might be expected that immunoconjugates of A chains would require a cleavable bond between antibody and hemitoxin to exhibit cytotoxicity.
Disulfide-bonded immunoconjugates were initially believed to be necessary to mimic the disulfide linkage of A and B chains of native toxin. This native disulfide bond had to be reductively cleaved to liberate the active A chain of the toxin molecule within the cell.
Linkage of A chains of toxins with antibodies through non-reducible bonds generally produced immunoconjugates of decreased potency. For instance, one early study reported that conjugates of polyclonal antibody and the A chain of diphtheria toxin, joined by a linker that did not contain a reducible bond, were one-third as active against target cells as conjugates linked with disulfide bond (Y. Masuho et al., Biochem. Biophys. Res. Comm. 102:561, 1981). This result was not surprising as optimum activity of diphtheria toxin requires limited proteolysis to allow reduction and release of the enzymatic portion.
Masuho et al. subsequently examined four ricin A chain immunoconjugates joined with different linkages (Y. Masuho et al., J. Biochem. 91:1583, 1982). Monovalent Fab'-SH fragments of polyclonal antibodies were cross-linked to A chain using 5,5'-dithio bis (2-nitrobenzoic aoid) to form disulfide bonds or N, N'-o-phenylenedimaleimide (PDM) to form thioether bonds. Reaction with PDM yielded essentially pure heterodimer (Fab'-PDM-A chain) without formation of the homodimer (Fab'-PDM-Fab'). Divalent F(ab').sub.2 fragments of the same polyclonal antibody were substituted with N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) or N-succinimidyl m-(N-maleimido) benzoate (SMB) prior to mixing with ricin A chain. In these reactions, the coupling reagents react with lysine residues of the F(ab').sub.2 fragment to form an amide bond; the sulfhydryl group of the A chain reacts with the activated disulfide bond of SPDP to form a disulfide bond between A chain and antibody or with the maleimide group of SMB to form a thioether bond. The PDM and SMB conjugates were resistant to cleavage with 2-mercaptoethanol (2-ME). Upon examination of the reaction products, the F(ab').sub.2 immunoconjugates were found to be substituted with 0, 1, 2, or 3 molecules of ricin A chain, with a 1:1 ratio of F(ab').sub.2: ricin A chain predominant.
Upon comparison, immunotoxins of polyclonal antibodies or antibody fragments conjugated to ricin A chain were 54-80 times more toxic when linked by a cleavable disulfide bond, rather than a noncleavable thioether or amide bond. The reduction in activity was not due to a blocking effect of the antibody or Fab' fragment on the enzymatic activity of the A chain, further suggesting that the A chain must be liberated from the cell binding moiety to exhibit cytotoxicity.
Masuho et al. also investigated the effects of antigen-binding valency on cytotoxic activity, and reported that divalent binding as with intact antibody or F(ab').sub.2, was superior to monovalent (Fab') binding. Since F(ab').sub.2 and Fab' fragments do not have an Fc region, which mediates non-antigen specific binding to cells, specificity of immunoconjugates should be improved in vivo.
Other investigators have examined the effects of cleavable and noncleavable linkers on the toxicity of A chain- and hemitoxin-containing conjugates (S. Ramakrishnan and L. Houston, Canc. Res. 44:201, 1984). Hemitoxins possess an enzyme activity functionally equivalent to that of A chain, but do not have an associated delivery polypeptide analogous to B chain. Pokeweed antiviral protein (PAP), a hemitoxin, does not contain native free thiol groups, and thus thiol groups need be introduced by reaction with an agent such as SPDP, followed by reduction. Reduction produces free sulfhydryl groups, with the average number of 1.24 per PAP molecule. Monoclonal antibodies (MAbs) directed against Thy 1.1 were subsequently derivatized with SPDP and conjugated with SPDP-PAP overnight in the cold using a 3-fold molar excess of PAP over IgG. The resulting immunoconjugate contained a cleavable disulfide bond.
Alternatively, a noncleavable immunoconjugate was formed by first reacting MAb with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), and then reacting the MAb-MBS protein with reduced SPDP-PAP. The formation of thioether bonds was performed at 4.degree. C overnight. Similar conjugates were formed with ricin A chain.
All immunoconjugates were highly specific for Thy 1.1-positive cells, however, variations in cytotoxicity were observed when different Thy 1.1 MAbs were incorporated into immunoconjugates. Ricin immunoconjugates (MAb 31-E6 and ricin A chain) joined by a noncleavable linker were effective inhibitors of in vitro translation, but were ineffective inhibitors of protein synthesis when incubated with intact target cells.
Unlike ricin A chain conjugates, PAP immunoconjugates (MAb 31-E6 and PAP) were as active with cleavable or non-cleavable linkers. These data indicated that PAP immunoconjugates do not require cleavage of the toxic moiety for inhibitory activity.
The observations of Ramakrishnan and Houston could not be confirmed in a subsequent study (J. Lambert et al., J. Biol. Chem. 260:12035, 1985). These investigators found that a cleavable linkage was required for cytotoxic activity of gelonin and PAP immunoconjugates, consistent with previous findings reported in the literature regarding the necessity of cleavage of A chain conjugates.
Recently, PE and ricin A chain immunoconjugates, linked with disulfide or thioether bonds, were assayed for human breast cancer cytotoxicity in vitro (M. Bjorn et al., Canc. Res. 46:3262, 1986). For preparation of disulfide-linked conjugates, PE was derivatized with 2-iminothiolane (IT). MAbs derivatized with SPDP were then reacted with PE-IT for 15-20 hours at 4.degree. C. to yield disulfide-bonded conjugates. Thioether-linked conjugates were formed by reacting MAbs with the maleimido-6-aminocaproyl ester of 4-hydroxy-3-nitrobenzene sulfonic acid prior to mixing with PE-IT in a 1:3 (MAb:PE) molar ratio for 15-20 hours at 4.degree. C. The resultant immunoconjugates were predominantly 1:2 (Ab to PE or ricin A chain).
The cytotoxicities of an analogous disulfide-linked and thioether-linked MAb-PE immunoconjugate were compared. The cleavable and noncleavable PE immunoconjugates displayed similar cytotoxic activities against two different target cells. In contrast, thioether-linked ricin A chain conjugates were less cytotoxic in vitro than the analogous disulfide-linked conjugates. The in vivo relevancy of these findings was unclear, since comparative in vivo studies were not performed. In a separate study, in vivo plasma clearance and stability of MAb-PE immunoconjugates in mice have been reported to be essentially the same for disulfide and thioether-linked immunoconjugates (L. Barbieri and F. Stirpe, Canc. Surv. 1:489, 1982).
Immunoconjugates of Pseudomonas exotoxin (PE) coupled to MAbs or to EGF through either disulfide or thioether linkages were disclosed in a recent patent (I. Pastan et al., U.S. Pat. No. 4,545,985, 1985). PE was treated with methyl-4-mercaptobutyrimidate (MMB), so as to introduce two thiol groups per PE molecule. MMB-PE was then reacted with dithiobis(2-nitrobenzoic acid) (DTNB), forming a PE derivative possessing disulfide bonds. MMB-derivatized MAb, which contains slightly more than 1 thiol group per MAb molecule, was then reacted with a 3-fold molar excess of DTNB-activated MMB-PE at room temperature for 2 hours. The conjugation of MMB-PE to MMB-EGF was performed in the presence of an excess of derivatized PE, and the reaction was allowed to go to completion. The patent postulated that MBS-modified MAb could be reacted with MMB-PE to yield a putative thioether-linked immunoconjugate. However, the production of MAb-PE immunoconjugates linked by thioether bonds was neither reduced to practice nor claimed. The reactants in this mixture could also yield disulfide-bonded PE-MMB-MAb, through reaction of MMB-PE with native disulfide bonds of MAb. Data comparing thioether- and disulfide-linked conjugates were not reported by Pastan et al.
The ratio of toxin:antibody present in immunoconjugates may also affect the specificity and cytotoxicity of the hybrid molecules. One report suggested that the cytotoxicity of holotoxin-immunoconjugates may be potentiated when immunoglobulin:ricin was in a ratio of 1:2, rather than 1:1. (J. Marsh and D. Neville, Jr., Biochem. 25:4461, 1986). Whole MAb thiolated with 2-iminothiolane and MBS-substituted ricin were combined in a 10:1 molar ratio of ricin:MAb. The yield of 1:1 species of immunoconjugates was reported to be in the range of 3%-8%. The authors compared conjugation with DTT-reduced MAb, which resulted in 10 -SH groups/antibody and concluded that it was desirable to introduce a limited number of thiol groups via heterobifunctional reagents into MAbs rather than using native sulfhydryl groups.
The literature reflects the historical development of approaches to immunoconjugate syntheses. Preliminary studies of the efficacy of "magic bullets" used reduced polyclonal antibodies linked to reduced toxins to form immunoconjugates. This method of conjugation was relatively uncontrolled and unpredictable, since antibody and ricin disulfide bonds could reform as readily as antibody-toxin hybrids. Heterobifunctional reagents became preferred compounds for linking antibody to toxin, because the amount of free sulfhydryl groups available for subsequent conjugation, and thus ratios of antibody to toxin in the immunoconjugate product, could be controlled. The presence of two different reactive end groups on heterobifunctional reagents permitted directed, predictable, and reproducible reaction of the linking agents.
A variety of reagents have been used in the literature to derivatize antibody or toxin molecules. However, most recently reported methods of producing immunoconjugates join derivatized toxin to derivatized antibody. U.S. Pat. No. 4,520,226 discloses MAb for immunotoxin production derivatized with MBS, but yields and in vivo utility were not reported. Most synthetic protocols generally combine antibody with a 3- to 10-fold molar excess of derivatized toxin. Thus, preparation of derivatized toxin represents a significant effort of labor and expense. Also removal of unreacted toxin presents a significant manufacturing problem.
Further, the conditions described in the art for conjugation of antibody and toxin often involve long reaction times. In addition, even with the relatively controlled conditions of conjugation presently used, the immunoconjugate products are often heterogeneous, and must be purified from unreacted components and undesired species, thereby significantly reducing the yield of the desired end product to thirty percent or less.
There are little data supporting the in vivo efficacy of immunoconjugate therapy. Most in vivo treatment has been disappointing due to (1) nonspecific toxicity of the immunoconjugates, which limits the amount of conjugate that can be administered; and (2) reduced delivery of toxin to the target site as a result of premature cleavage of the disulfide linkage in vivo, or binding to receptors in normal tissues, e.g., liver. In the latter case, premature release of toxins, especially holotoxins, can greatly increase nonspecific toxicity. Cocktails of immunotoxins have been explored for increasing efficacy of treatment ex vivo. Donor bone marrow cells were treated ex vivo with immunoconjugates cytotoxic for T-cells, prior to infusion into patients, for treatment of GVHD (D. Neville, Jr. and R. Youle, U.S. Pat. No. 4,520,226, 1985). The combination of three different anti T-cell conjugates improved T-cell depletion compared to single antibodies. This procedure has not yet been tested for in vivo application.
As a result of the above-noted disadvantages of current immunoconjugates, there is a need in the art for improved immunoconjugates that can be efficiently and rapidly synthesized in high yield and that exhibit reduced nonspecific toxicity in vivo. Conjugates possessing these properties will permit administration of an effective in vivo therapeutic dose of an immunoconjugate that is delivered efficiently to targeted tumor sites. The method of conjugation should conserve toxin by minimizing the ratio of toxin offered to antibody. Finally, such conjugates should be linked through a more stable thioether bond, rather than less stable disulfide bonds, using native sulfhydryl groups in the antibody or toxin. The present invention fulfills these needs, and further provides other related advantages.